Complex-bound inhibitors of metabolic enzymes capable of being activated, useful as molecular markers for diagnostic and therapy monitoring purposes

ABSTRACT

This invention relates to the use of an inhibitor of an activatable metabolic enzyme, which inhibitor is bound to a high molecular weight carrier, as a molecular marker for determining the activation of this enzyme for the diagnosis of the enzyme. The invention relates in particular to the use of a thrombin inhibitor, which is bound to a high molecular weight carrier, as a molecular marker for determining clotting activation in clotting diagnosis and therapy monitoring. The invention preferably relates to the use of dextran-hirudin or PEG-coupled hirudin as a molecular marker for clotting diagnosis and therapy monitoring.

The invention relates to the use of inhibitors of activatable oractivated metabolic enzymes, which inhibitors are bound to highmolecular weight carriers, as molecular markers for determining theactivation of the enzyme in order to indirectly diagnostically andtherapeutically monitor the enzyme.

This invention in particular relates to the use of an inhibitor of anactivation product of the blood clotting cascade or of an activatedfibrinolysis enzyme, which inhibitor is bound to a high molecular weightcarrier, as a molecular marker to indirectly monitor an activationproduct of the blood clotting cascade or an activated fibrinolysisenzyme. The invention preferably relates to the use of a thrombininhibitor, which is bound to a high molecular weight carrier, as amolecular marker for determining clotting activation in clottingdiagnosis and therapy monitoring. The invention relates in particular tocomplex-bound hirudin (CBH) as a molecular marker for determiningclotting activation.

Many metabolic physiological processes are regulated via cascademechanisms, which are often branched, and in which an initiator or achain reaction is intensified via a series of activatable enzymes. Forexample, mechanisms such as these are effective in the regulation ofglycogen metabolism, in the transmission of extracellular signals and inblood clotting in particular. On a molecular level, sequentialphosphorylation of the factors involved in a cascade mechanism is oftento be found here. The intensification of an initiator by this mechanismis due to the enzymes which participate in the different stages of themechanism being capable of modifying a plurality of substrate molecules,which themselves are often enzymes also. The activated enzymes of amechanism such as this are suitable as molecular markers for determiningthe activation reaction which is proceeding in the body in each case. Ina procedure such as this, specific, high-affinity inhibitors of therespective activated key enzyme are used which are to be fed as such toa permanent diagnosis line. This so-called molecular marker principlehas only been developed unsatisfactorily hitherto, however.

The search for suitable molecular markers for the determination ofintravasal activation reactions of blood clotting has been intensivelypursued for some years. In the course of this work a series of metabolicproducts of clotting activation has emerged as molecular markers. Thesecomprise prothrombin F1+2 fragments, platelet factor IV andfibrinopeptides A and B, as well as some cleavage products of fibrindecomposition (e.g. d-dimers) which are produced by fibrinolysis, andalso comprise complexes formed between natural antithrombins and theserine protease thrombin, which are known as TAT complexes.

The usability of these molecular markers has been analysed inlarge-scale clinical studies. It has generally been ascertained that itis possible to determine blood levels of molecular markers of this typewhich are definitely increased or which persist during thromboticoccurrences or thrombo-embolytic diseases. The efficiency of thesemarkers leaves very much to be desired, however. In clinical patientswho suffered from venous thromboses or from arterial thromboticocclusion diseases, the response capacity of the most sensitive markerfor clotting, namely prothrombin fragment F1+2, was less than 20%.Moreover, no correlation could be found between the severity of thethrombo-embolytic disease or thrombotic occurrence and the magnitude ofthe blood level of this and other molecular markers.

From experience with persons suffering from diseases of this type, itcan be deduced that in haemostaseology there has hitherto not been aprinciple of molecular measurement which enables conclusions to be drawnon the intensity of clotting activation by determining the actual bloodlevel of the marker. The cause of this is that the molecular markers, asmetabolic products of clotting enzymes which occur naturally in thebody, are removed more or less rapidly from the circulation byelimination mechanisms. In this respect, it has to be taken intoconsideration that the markers are metabolised more or less rapidly,depending on the function of the organ concerned, particularly in thearea of liver metabolism.

The underlying object of the present invention is thus to identifysensitive markers for the detection of an early phase of metabolicactivation.

More particularly, an underlying object of the present invention is thusto identify sensitive markers for the detection of an early phase ofclotting activation.

Moreover, the molecular marker should have a wide diagnostic range forthe determination of intravasal activation reactions and morespecifically for determining blood clotting reactions. In this respect,the marker should act in the organism independently of metabolicprocesses or elimination reactions. It must be ensured that the markeris distributed rapidly, exclusively in the blood circulation, is notmetabolised or is only metabolised to a slight extent, and is onlyeliminated slowly.

This object is achieved by the use of a specific inhibitor of an enzymeinvolved in metabolism, which inhibitor is bound to a high molecularweight carrier, and thereby functions as an indirect marker formonitoring the activation or activity of this enzyme, e.g., an enzymeinvolved in the blood clotting cascade or fibrinolysis.

Surprisingly, it has now been found that inhibitors of an activatablemetabolic enzyme, which inhibitors are bound to a high molecular weightcarrier, i.e. complex-bound inhibitors of an activation product of theblood clotting cascade or of an activated fibrinolysis enzyme, areexclusively and rapidly distributed in the blood circulation, are onlydecomposed or eliminated slowly in the organism, and still have almostthe same affinity for the enzyme as do the free, un-bound inhibitors.This principle can be employed for all activatable key enzymes for whichhigh-affinity and specific inhibitors are available. It is particularlysuitable for the activation products of the clotting cascade, such asthrombin, activated factor VII or activated factor X, and is alsosuitable for activated fibrinolysis enzymes, e.g. tissue plasminogenactivator (tPA) or plasmin. Examples of other key enzymes involved inmetabolism which can be inhibited by high-affinity specific inhibitorsinclude angiotensin-converting enzyme (involved in blood pressureregulation) and elastase (involved in shock reactions).

The description given below relates to a preferred embodiment of theinvention, namely the use of thrombin inhibitors. However, it should beunderstood that the molecular marker principle which is illustrated inthis example for the diagnostic monitoring of blood can be employedcorrespondingly for any combination of an activatable enzyme and ahigh-affinity, specific inhibitor, which is bound to a high molecularweight carrier substance.

It has surprisingly been found that thrombin inhibitors which are boundto high molecular weight carriers (complex-bound thrombin inhibitors)are exclusively and rapidly distributed in the blood circulation, areonly decomposed or eliminated slowly in the organism, and also alwayshave the same affinity for thrombin as do free thrombin inhibitors andare thereby suitable for diagnostic purposes. It is not possible to useun-bound thrombin inhibitors for diagnoses of this type, because thesesubstances are distributed in the body as a whole, not only in theblood, and when consumed they become redistributed without a detectabledecrease in their concentration in the blood.

High-affinity natural thrombin inhibitors. e.g. hirudin, and also allother direct-binding synthetic thrombin inhibitors which have a highaffinity for thrombin, can be used as thrombin inhibitors. Examplesthereof include PEG-bonded 4-amidinophenylalanine (see Peptide Research8, No. 2, 78-85 (1995)). Natural or synthetic substances can be used asthe high molecular weight carriers. Examples include polyethyleneglycol, dextran, and also blood proteins which occur naturally in thebody. Other examples thereof include albumin, γ-globulins, and alsoferritin, succinylated gelatine, crosslinked polypeptides, andpolyhydroxy-starch.

A dextran-bound (DP) hirudin is suitable for use, as is hirudin which iscoupled to polyethylene glycol (PEG), or hirudin which has been bound tohuman body proteins. Due to their molecular size, these proteins(albumin-bound hirudin or hirudins which are bound to definedgamma-globulins) only undergo a very slow biological eliminationprocess.

Complex-bound hirudins of this type have the considerable advantage thatthey are distributed almost exclusively in the blood and exhibit noextravasation into the extracellular fluid space. Even small amounts ofthis marker, when distributed in the blood, can thereby function as ahigh-activity inhibitor, which binds rapidly and strongly, forintravasally activated thrombin or activated intermediates of theprothrombin-thrombin transformation. Due to the binding of the activatedenzyme to the complex-bound thrombin inhibitor, the amount of "free"complexed hirudin is reduced by the extent to which active thrombinbecomes available in the circulation.

Hirudin, and also hirudin which is bound to macromolecules, has highaffinity for the serine protease thrombin. Its singular specificityexclusively for thrombin species allows this marker only to becomeactive for this serine protease in the organism. Binding to otherenzymes is not possible and is not known.

If the key enzyme of the clotting system, namely the serine proteasethrombin, becomes available in the blood in the organism due to apermanent clotting activation, as the final product of intrinsic orextrinsic clotting, it is immediately bound and deactivated by the CBHmolecular marker which is present in the blood. The amount ofcomplex-bound hirudin, namely of the free inhibitor, decreases inflowing blood by the extent to which thrombin-hirudin complexes areformed.

The "free" molecular marker, namely complex-bound hirudin, is determinedwith the aid of a sensitive, specific method of detection for freehirudin which can rapidly be carried out. With the aid of this method ofdetection, discrete clotting activation effects can also be detected andquantified at an earlier point in time in the organism by repeatedmonitoring.

The molecular markers according to the invention are employed at a doseof 0.005-0.5 mg/kg, preferably 0.01-0.05 mg/kg, most preferably0.01-0.02 mg/kg, with respect to the body weight of the patient. Theyare administered parenterally, preferably intravenously. Oralapplication, in which resorption of the marker is ensured by acorresponding formulation, is also possible. For this purpose, themolecular markers according to the invention are employed, together withcustomary adjuvant substances and carrier substances, in a formulationwhich is suitable for this method of administration. Thus, for example,complex-bound hirudin preparations can be produced either in afreeze-dried formulation (to be dissolved in 5 ml water; PEG-hirudin,albumin-hirudin, γ-globulin-hirudin) or as a ready-to-use injectionsolution (e.g. 5 ml; gelatine-hirudin, hydroxy-starch-hirudin), whereinthe content of hirudin is appropriately 5 mg hirudin/ampoule. Therequisite dose for the application is calculated according to theformula

kg body weight: 20= ml application volume and is administered as anintravenous bolus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of continuous thrombin infusion on the plasmalevel of PEG-hirudin/144 (140 U/kg).

FIG. 2 shows the effect of brief and repeated thrombin infusions (3×20minutes) on the plasma level of PEG-hirudin /153 (150 U/kg).

FIG. 3 shows the effect of thrombokinase infusion on the blood level ofdextran-hirudin.

The invention is explained in more detail with reference to thefollowing example.

Example: Complex-bound Hirudin as a Molecular Marker

Patients from the group at risk were given an intravenously administereddose of complex-bound hirudin corresponding to their body weight. Aftera short distribution period (10-15 minutes) a constant blood level ofthis CBH was reached. The elimination half-life for CBH was known as acomparison quantity from corresponding control investigations on healthytest volunteers.

Small amounts of citrate whole blood (0.5 ml) were taken at short timeintervals from patients at risk from thrombosis. The free circulatingCBH was determined with the aid of a specific detection method forhirudin, namely the ecarin clotting time (ECT, European Patent No. 93903 232.2). The ecarin clotting time is a method of determining activityand is extremely sensitive for the detection of free hirudin, but notfor hirudin-thrombin complexes. There was a direct correlation betweenthe decrease in the blood level of CBH and the intensity of thrombinliberation in the blood circulation. A quantification of the liberationof thrombin in the bloodstream with time was obtained due to thepossibility of mathematically modelling the "rate of disappearance" orthe more pronounced decrease in the blood level of free CBH. By usingthis method, it is even possible to detect early phases of clottingactivation, which could not hitherto be achieved diagnostically.

In further tests, these molecular thrombin "probes" were checked fortheir efficiency in a modelling study applied to experiments on animals.

The experimental animals used here were rats (HAN-WISt, CentralExperimental Animal Unit of the University of Jena) and rabbits(Chinchilla bastards, Savo, Bad Kislegg). Both male and female animals,conforming to the SPF livestock standard, were used from both species.The rats were narcotised with ethylurethane (1.5 g/kg subcutaneously),and the rabbits were narcotised with pentobarbital (25 mg/kgintravenously). Two different methods of detection were used as methodsof determining hirudins in blood. In addition to the ecarin clottingtime, a chromogenic substrate method was also used. In this method,chromogenic substrate (Chromozym TM, Pentapharm Basle) and ecarin (25Eu/ml) or thrombin (1.5 NIH U/ml) were added to the plasma (diluted1:10), and the extinction was measured in a spectrophotometer after twominutes (NIH U: International Standard for Thrombin Clotting Activity;NIH=National Institute of Health).

The complex-bound hirudin was used in two different hirudinpreparations:

1. Dextran-hirudin (Dextran 150 kDa) was bound to hirudin by means of amethod according to Walsmann et al. The specific activity was 971 ATU/mg(ATU=anti-thrombin units=International Standard for Direct ThrombinInhibitors).

2. PEG-coupled hirudin. Commercial preparations supplied by Knoll wereused (PEG-hirudin/144 or PEG/153).

1. Effect of Thrombin on the Plasma Level of PEG Hirudin in Rats

Experimental Design:

Narcotised rats were given PEG-hirudin in a dosage of 140 ATU/kg bodyweight, administered intravenously as a bolus (ATU=anti-thrombin units).10 minutes later the rats were given an infusion of thrombin and thecontrol animals were given the same infusion volume of saline. 0.5 ml ofcitrate blood was taken from the rats at 60 minute intervals via apermanent catheter in the jugular vein, so as thereby to monitor theblood level of the molecular marker. Thrombin was infused into the ratsin the following concentrations over 360 minutes: 35, 70, 140, 250 and500 NIH U/kg×hour⁻¹. The results are illustrated in FIG. 1. It can beseen that after a short initial distribution period a relativelyconstant blood level of PEG-hirudin was detected in the plasma. Theapproximate half-lives were about 12-14 hours. For an infusion of 35 NIHU/kg×hour⁻¹ thrombin, the time-dependence of the blood level ofPEG-hirudin was almost identical to that of the control group (salineinfusion). At 70 NIH U/kg×hour⁻¹ thrombin, a significantly accelerateddecrease in the blood level of PEG-hirudin was detected even after 120minutes. At higher doses of thrombin, free PEG-hirudin disappeared veryrapidly from the blood circulation. At 250 or 500 NIH U thrombin,PEG-hirudin could no longer be detected in the plasma even after 120minutes.

2. Effect of Repeated Thrombin Application on the PEG-hirudin Level ofRats

The PEG-hirudin (PEG153) which was used in these tests exhibited atime-dependence of its distribution and blood level which was almost thesame as that in the test example described above. After a brief 15minute infusion of 100 NIH U/kg thrombin in each case, only a discreteinfluencing of the PEG-hirudin level was detected after 120, 180 and 240minutes, whereas on the application of 250 NIH-U/kg thrombin a morepronounced decrease in the PEG-hirudin blood level occurred after 3hours; on increasing the dose to 500 NIH U/kg thrombin, this decreasewas even greater.

3. Effect of Thrombokinase on the Plasma Level of Dextran-hirudin inRabbits

The rabbits were pre-treated with 5000 ATU/kg of dextran-hirudin. Fromthe monitoring of the dextran-hirudin blood level it could be seen thata constant hirudin blood level in the rabbits first detected after 24hours. On the infusion of a purified thrombokinase solution (1ml/kg/hour) over 6 hours, a more pronounced decrease in thedextran-hirudin blood level was detected. For these investigations, theresults of five separate tests were combined.

It follows from these investigations that dextran-hirudin has a verylong distribution phase (24 hours) in rabbits, due to the interactionsof the dextran with surface structures of the endothelium cells, withthe RES of the liver and with the corpuscular constituents of the blood.For this species of animal, dextran-hirudin was only of limitedsuitability for marker investigations. It could not be identified fromthese investigations whether the dextran-hirudin was also distributed indeeper compartments of the rabbit organism. In contrast, the PEG-coupledhirudins (PEG 144 and 153) proved to be suitable for correspondingmolecular marker modelling in the tests on rats which are presentedhere. A relatively constant distribution equilibrium was attained in thecirculation of rats, even after 10 minutes, and modelling of intravasalclotting activation by means of the continuous infusion of small amountsof thrombin, or by the discontinuous application of thrombin, could befollowed and measured by a corresponding disturbance of the PEG-hirudinblood level.

It can be deduced from these modelling investigations of animalexperiments that complex-bound hirudins are suitable as molecularmarkers for intravasal clotting activation. The advantage of themolecular markers which are presented here is that high molecular weighthirudin complexes of this type are not subject to any eliminationfunction in the organism. They are permanently available for bindingactive thrombin, which can occur permanently in the circulation as afinal activation product of clotting activation. A definite clottingactivation can be quantified by a sensitive method of detecting thelevel of free marker.

We claim:
 1. An method for indirectly quantitating a known enzyme, comprising the following steps:(1) parenterally administering to a subject in need of such monitoring an amount of an inhibitor-high molecular weight complex or inhibitor-high molecular weight conjugate, which inhibitor-high molecular weight complex or conjugate specifically binds to said enzyme to produce a complex; (2) measuring the change in the circulating amount of said inhibitor-high molecular weight conjugate or complex in the blood of said subject after administration; and (3) correlating said change to the amount of said enzyme.
 2. The method of claim 1, wherein the enzyme is one known to be involved in one of (i) a reaction in the clotting cascade, (ii) fibrinolysis, (iii) a metabolic physiological process, and (iv) shock.
 3. The method of claim 1, wherein said enzyme is selected from the group consisting of thrombin, factor VII, factor X, tissue plasminogen activator (tPA), plasmin, angiotensin-converting enzyme and elastase.
 4. The method of claim 1, wherein the inhibitor is a thrombin inhibitor.
 5. The method of claim 4 wherein said thrombin inhibitor is hirudin and the enzyme which is indirectly determined is thrombin.
 6. The method of claim 1, which is used to indirectly measure thrombin in order to monitor clotting function.
 7. The method of claim 8 wherein said inhibitor is hirudin.
 8. The method of claim 1, wherein the high molecular weight compound is selected from the group consisting of dextran, polyethylene glycol (PEG), albumin, gamma-globulin, ferritin, succinylated gelatin and polyhydroxy-starch.
 9. The method of claim 1, wherein the enzyme which is indirectly monitored is one that catalyzes a fibrinolytic reaction.
 10. The method of claim 1, wherein the enzyme which is indirectly monitored is one that catalyzes a reaction in the clotting cascade.
 11. The method of claim 1, wherein the inhibitor-high molecular weight compound conjugate or complex is dextran-hirudin or PEG-hirudin.
 12. The method of claim 1, wherein the amount of said inhibitor-complex or conjugate administered to said subject ranges from 0.005 to 0.5 milligrams per kilogram.
 13. The method of claim 12 wherein said amount ranges from 0.01 to 0.05 milligrams per kilogram.
 14. The method of claim 13 wherein said amount ranges from 0.01 to 0.02 milligrams per kilogram. 